The goal of this proposal is to define the regulatory mechanisms that control the bacterial cell cycle and to understand how these mechanisms function within an integrated system. We have shown that Caulobacter exerts exquisite spatial and temporal control of its cell cycle by the use of transcriptional and proteolytic networks integrated with dynamic subcellular protein localization. Key to cell cycle control, a small number of master transcriptional regulators orchestrate cell cycle progression. A two component phospho-signaling pathway, involving the polarly-localized CckA histidine kinase, mediates the activation of the CtrA master regulator whose function is to regulate the genes involved in polar morphogenesis and the biogenesis of the cell division apparatus. Using robotic high throughput screens for genes involved in protein localization, we identified the DivL kinase for the localization of the CckA histidine kinase, and the CpaE pili protein for the localization of the PleC histidine kinase that is essential for polar morphogenesis. We will explore the mechanism of polar localization of these critical kinases and determine how it is related to their function within the cell cycle regulatory circuit. To understand the cell cycle integration of transcriptional regulation, we will define the mechanism of action of the newly identified SciP transcriptional regulator that functions as a repressor of CtrA activated genes at a specific time in the cell cycle, and the novel CrfA non-coding RNA that modifies the cell cycle regulatory circuitry in response to nutrient deprivation. Finally, the mid-cell establishment of the FtsZ cytokinetic ring is dependent on signals from the cell poles and is an integral component of the core cell cycle circuitry. Accordingly, we will explore the temporally regulated localization, assembly, and disassembly of the divisome, as a function of the cell cycle. PUBLIC HEALTH RELEVANCE: We examine the molecular mechanisms of each of the consecutive steps in the bacterial cell cycle and then elucidate how these individual events are integrated into a functional system. This approach has allowed the identification of novel mechanisms that coordinate the temporal and spatial control of cell cycle progression, leading to the identification of new antibiotic targets and ultimately the design and development of a new class of antibiotics.